The pharmaceutical industry today faces two fundamental challenges in its drug development to process, namely the identification of appropriate protein targets for disease intervention and the identification of high quality drug candidates which act specifically on these targets. These two challenges are of paramount importance in the design of successful medicines.
Intervention with low-molecular-weight compounds represents a fundamental therapeutic concept for the treatment of human disorders. Due to their key roles in signal transduction processes implicated in the onset and progression of severe diseases such as human cancers, various members of the protein kinase superfamily of enzymes have been extensively targeted by small molecules disrupting their catalytic functions. These drug development efforts have provided a plethora of tools for the dissection of cellular signalling by chemical genetic approaches. In contrast to classical genetic inactivation, small molecule inhibition can selectively modulate the catalytic activity of a protein kinase in a way that is rapid, tunable and, in most cases, reversible. Moreover, many protein-protein interactions formed by protein kinases are preserved in the presence of small molecule antagonists and can therefore be dissected from their catalytic functions.
Despite these obvious advantages, small molecule antagonists developed for kinase inhibition have the potential to inactivate several targets in intact cells, due to common structural features found in the catalytic domains of different protein kinases or even members from other enzyme families. The selectivity of kinase inhibitors can be assessed by parallel in vitro activity or binding assays for large numbers of recombinant protein kinases (Fabian, M. A. et al., Nat. Biotechnol. 23, 329-36 (2005); Davies, S. P. et al., Biochem. J. 351, 95-105 (2000)). This approach undoubtedly provides valuable and quantitative data about drug selectivity, but has two major limitations: First, in addition to a significant part of the protein kinase complement (=the kinome), potential targets from other enzyme classes are underrepresented or missing altogether in these screening formats. Secondly, and perhaps more importantly, the recombinant kinase collection included in a selectivity panel does not match the cellular profile of potential targets expressed in, for example, a cell system where the biological effects of a kinase inhibitor are investigated.
These shortcomings can be addressed with proteomic approaches, which employ immobilized compounds, e.g., kinase inhibitors, for the selective affinity purification of target proteins in combination with protein identification, e.g., by mass spectrometry (MS). This straightforward technique has been successfully used to identify the target components of various kinase inhibitors in cellular extracts. However, target component identification was limited in the sense that the affinities of the inhibitor towards its cellular binding partners could not be inferred from the MS data. For obtaining further quantitative information, it was necessary to resort to secondary, in vitro activity assays to identify those inhibitor targets, which were potently inhibited and therefore potentially relevant for the observed cellular drug actions. However, in practice, it is challenging to assess the whole target spectrum of a small molecule drug due to the fact that the required recombinant proteins are not all available or in vitro activity assays prove difficult to establish.
Comprehensive knowledge of the cellular proteins targeted by small molecule intervention is a pre-requisite to define chemical-biological interactions on the molecular level (Daub, H. et al., Assay Drug Dev. Technol. 2, 215-24 (2004); Fabian, M. A. et al., Nat. Biotechnol. 23, 329-36 (2005)). Although affinity purification techniques together with mass spectrometry (MS) have been successfully used to identify the interacting proteins of immobilized small molecule inhibitors, these previous proteomics approaches did not deliver information of cellular target affinities (Godl, K. et al., PNAS U.S.A., 100, 15434-9 (2003); Brehmer, D. et al., Cancer Res. 65, 379-82 (2005); Daub, H., Biochim. Biophys. Acta 1754, 183-90 (2005)).
There is therefore a need for methods of proteomics which permit the direct qualitative and/or quantitative evaluation or determination of compound-target component interactions, e.g., inhibitor-target protein interactions. There is also a need for methods of the afore-mentioned kind which can be adapted for high throughput applications.